产N乙酰神经氨酸重组大肠杆菌的构建及其生物转化合成
发布时间：2018-07-17 06:04
【摘要】：唾液酸是一类含有9个碳原子并具有吡喃糖结构的酸性氨基糖,又称神经氨酸;唾液酸在自然界中分布广泛,且种类繁多,迄今已发现超过60种唾液酸。N-乙酰神经氨酸(Neu5Ac)是最重要的一种唾液酸,是其他唾液酸合成的前体物质,与人类的健康关系最为密切;Neu5Ac可以用于合成抗病毒药物,治疗H1N1和H5N1流感等疾病。此外,Neu5Ac也有很大的营养价值,它能促进婴儿的大脑发育,同时对维持早产儿脑功能和健康也有积极作用。目前制备和生产Neu5Ac常用的方法是酶催化法,该方法产量虽高,但需要添加过量的丙酮酸导致原料浪费、产品纯化困难以及环境压力增大。本论文尝试建立一个不依赖丙酮酸的Neu5Ac合成方法:以N-乙酰葡萄糖胺(Glc NAc)为底物,在N-乙酰葡萄糖胺异构酶(AGE)的催化下异构化为N-乙酰甘露糖胺(Man NAc);然后,在Neu5Ac合成酶(Neu B)的催化下,与磷酸烯醇式丙酮酸(PEP)发生不可逆反应合成Neu5Ac。在大肠杆菌中构建上述Neu5Ac合成途径,并利用代谢工程和发酵工程方法强化Neu5Ac的合成。主要结论如下:(1)分别考察不同来源的AGE和Neu B的活性及酶学性质,对比得到在大肠杆菌中表达活性最高的酶。结果表明,猪肾脏中p AGE和项圈藻中b AGE的稳定性、温度偏好和p H偏好性相似,其最适p H均为7.0,最适温度均为50°C,并且在35-55°C范围内保持较高活性,达到其在50°C时活性的90%以上。在37°C Tris-HCl(p H 7.0)条件下,p AGE和b AGE活性分别为392.1和2411.5 U?mg-1总蛋白。此外,e Neu B和c Neu B的最适p H均为中性,且最适温度均为45°C,在30-47.5°C范围内保留超过60%的活性。在37°C Tris-HCl(p H 7.0)条件下,e Neu B和c Neu B活性分别为0.037和6.380 U?mg-1总蛋白。而c Neu B的稳定性不佳,2 h后仅有50%的催化活性保留。当与分子伴侣质粒p Gro7共表达时,测得的c Neu B活性比对照组提高17.8%;其诱导表达的Gro EL/Gro ES复合体,能阻止蛋白质的错误聚集并提高蛋白质的折叠速度,提高大肠杆菌可溶性表达c Neu B的能力。因此考虑基于b AGE和c Neu B两种酶构建Neu5Ac合成途径。(2)基于b AGE和c Neu B,构建Neu5Ac合成途径;分别敲除宿主菌的Neu5Ac分解途径和Glc NAc跨膜转运途径的相关基因,促进Neu5Ac的合成。由于c Neu B活性远小于b AGE,因此重点关注体系中的c Neu B催化活性。对调两个基因的次序,分别得到共表达载体p DTrc-AB和p DTrc-BA,发现p DTrc-AB中cneu B基因位于下游其表达活性更高。宿主大肠杆菌中的Neu5Ac分解途径保留时,无法实现Neu5Ac的积累;利用Red重组系统敲除nan ATEK基因簇后,得到E.coli SA-01,其Neu5Ac分解途径被阻断;将共表达载体p DTrc-AB和p DTrc-BA分别转化E.coli SA-01,经诱导后的菌体成功实现Neu5Ac的合成,Neu5Ac合成量分别为2.61和2.03 g?L-1。改造Glc NAc跨膜转运途径,避免底物Glc NAc与PEP在旁路上的无效消耗,增加胞内底物Glc NAc浓度和PEP供给,强化了Neu5Ac的合成,Neu5Ac合成量提高38.3%,达到3.61 g?L-1。(3)将大肠杆菌中PEP合成相关基因单独或共同过表达,提高胞内PEP的供给,进一步促进了Neu5Ac的合成。从E.coli MG1655基因组中克隆得到PEP合成相关基因pck和pps A,利用Duet系列载体构建两个基因的单独过表达和共过表达载体,并导入DE3溶源化的E.coli。当pck和pps A单独过表达时,Neu5Ac合成量分别为5.80和7.09 g?L-1。当pck和pps A共同过表达时,两个基因的转录水平同步提高,并正相关于载体的拷贝数。最优的PEP强化供给系统由中拷贝载体p CDF-pck-pps A构成,此时Neu5Ac合成量达到8.63 g?L-1,比出发菌株提高139%。(4)考察生物转化阶段合成Neu5Ac的营养条件以及表面活性剂对Neu5Ac合成的影响,优化Neu5Ac合成过程,并在发酵罐中实现Neu5Ac的高效合成。在含有氮源的培养基中,宿主大肠杆菌大量生长,而Neu5Ac的合成受到抑制;在无氮葡萄糖培养基中,宿主大肠杆菌生长受到限制,而Neu5Ac的合成抑制解除,合成量达到8.61 g?L-1。当宿主大肠杆菌生长旺盛时会消耗PEP,与Neu5Ac合成竞争胞内的PEP,反而不利于Neu5Ac的合成。当甘油为碳源时,其跨膜转运不依赖于PTS,节省胞内PEP从而使其更多流向Neu5Ac合成方向,Neu5Ac合成量也随之提高到10.43 g?L-1。转化培养基中添加Triton X-100,对Neu5Ac合成有一定的促进作用,但仅为6.1%。最后,在发酵罐中进行生物转化合成Neu5Ac过程,培养基中磷酸盐浓度大幅降低,减小了反应体系渗透压力;同时发酵罐中更好的传质状况,有利于Neu5Ac的合成。在发酵罐中65 h合成Neu5Ac达到16.02 g?L-1,比同条件摇瓶水平显著增加53.6%,实现了Neu5Ac的高效合成。
[Abstract]:Sialic acid is a class of acid aminosaccharides containing 9 carbon atoms and with the structure of Piran sugar, also known as neuraminic acid; sialic acid is widely distributed in nature and has a wide variety. Up to now, more than 60 sialic acid.N- acetyl neuroammonia acid (Neu5Ac) is the most important sialic acid, the precursor of other sialic acid synthesis, and human The health relationship is most closely related; Neu5Ac can be used to synthesize antiviral drugs and to treat diseases such as H1N1 and H5N1 influenza. In addition, Neu5Ac also has great nutritional value. It can promote the development of the brain in infants, and also have a positive effect on the maintenance of brain function and health in premature infants. At present, the method of preparing and producing Neu5Ac is enzyme catalysis, which is a common method. Although the yield of the method is high, it needs to add excess pyruvic acid to lead to waste of raw materials, difficult product purification and environmental pressure. This paper attempts to establish a Neu5Ac synthesis method without pyruvic acid: N- acetyl glucosamine (Glc NAc) as the substrate and the N- acetyl glucosamine isomerase (AGE) catalyzed isomerization to N- acetyl mannose Amines (Man NAc); then, under the catalysis of Neu5Ac synthetase (Neu B), an irreversible reaction with phosphoenolpyruvic acid (PEP) is used to synthesize Neu5Ac. in Escherichia coli to construct the above-mentioned Neu5Ac synthesis pathway, and to strengthen the aggregation of Neu5Ac by metabolic engineering and fermentation engineering. The main conclusions are as follows: (1) examine AGE and Neu B from different sources, respectively. The results showed that the stability of B AGE in P AGE and colipora colipora was similar in the pig kidney, and the temperature preference and P H preference were similar. The optimum P H was 7, the optimum temperature was 50 degrees C, and the high activity was maintained in the 35-55 degree C peri, which reached 90 of its activity at 50 degree C. Under the condition of 37 C Tris-HCl (P H 7), P AGE and B AGE activity are 392.1 and 2411.5 U mg-1 total protein respectively. And the total protein of 6.380 U? Mg-1. But the stability of C Neu B was not good, only 50% of the catalytic activity was retained after 2 h. When co expression with the molecular chaperone P Gro7, the B activity of C Neu was 17.8% higher than that of the control group. Enterobacteriaceae expressed the ability to express C Neu B. Therefore, we consider the construction of Neu5Ac synthesis pathway based on the two enzymes of B AGE and C Neu B. (2) the synthesis pathway is constructed based on B AGE and C. Therefore, we focus on the catalytic activity of C Neu B in the system. A co expression vector p DTrc-AB and P DTrc-BA are obtained in order to adjust the two genes. It is found that cneu B gene in the P DTrc-AB is more active downstream, and the accumulation of the Neu5Ac decomposition pathway in the host Escherichia coli can not be realized. After the Nan ATEK gene cluster, E.coli SA-01 was obtained, and its Neu5Ac decomposition pathway was blocked; the co expression vector p DTrc-AB and P DTrc-BA were converted to E.coli SA-01. The induced organisms were successfully synthesized, and the synthesis amount was 2.61 and 2.03 respectively. Ineffective consumption, increasing the concentration of intracellular substrate Glc NAc and PEP supply, enhanced the synthesis of Neu5Ac, increased the synthesis of Neu5Ac by 38.3%, reached 3.61 G? L-1. (3), by which PEP synthesis related genes in Escherichia coli were expressed individually or together, improving the supply of intracellular PEP and further promoting the synthesis of Neu5Ac. Related genes PCK and PPS A, using Duet series vectors to construct a single overexpression and co expression vector of two genes, and import DE3 soluble E.coli. when PCK and PPS A are expressed separately, the Neu5Ac synthesis is 5.80 and 7.09 g, and the transcriptional level of the two genes increases synchronously and is positively correlated. The number of copies of the carrier. The optimal PEP fortified supply system is composed of medium copy carrier P CDF-pck-pps A. At this time, the Neu5Ac synthesis amount to 8.63 G? L-1, increase 139%. (4) compared with the starting strain, investigate the nutritional conditions of the synthesis of Neu5Ac and the effect of surfactant on Neu5Ac, optimize the Neu5Ac synthesis process, and in the fermenting tank. To achieve high efficiency synthesis of Neu5Ac. In the medium containing nitrogen sources, the host Escherichia coli grew in large quantities, and the synthesis of Neu5Ac was inhibited. In the nitrogen free glucose medium, the growth of Escherichia coli in the host was restricted, and the synthesis of Neu5Ac was inhibited and the synthesis reached 8.61 G? L-1. when the host Escherichia coli grew exuberant PEP and N The synthesis of PEP in the competitive cell of eu5Ac is not conducive to the synthesis of Neu5Ac. When glycerol is a carbon source, its transmembrane transport is not dependent on PTS, which saves the intracellular PEP and makes it more flow to the direction of Neu5Ac synthesis. The Neu5Ac synthesis also increases to the 10.43 G? L-1. conversion medium, adding Triton X-100, but has a certain promotion effect on the Neu5Ac synthesis. In the end of 6.1%., the process of biosynthesis of Neu5Ac in the fermenting tank was carried out. The phosphate concentration in the medium was greatly reduced and the permeation pressure of the reaction system was reduced. At the same time, the better mass transfer in the fermenting tank was beneficial to the synthesis of Neu5Ac. In the fermenting tank, the 65 h synthesis Neu5Ac reached 16.02 G? L-1, which was significantly increased by 53.6% than that of the same condition shake flask. The efficient synthesis of Neu5Ac is realized.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ920.1
本文编号：2129334
[Abstract]:Sialic acid is a class of acid aminosaccharides containing 9 carbon atoms and with the structure of Piran sugar, also known as neuraminic acid; sialic acid is widely distributed in nature and has a wide variety. Up to now, more than 60 sialic acid.N- acetyl neuroammonia acid (Neu5Ac) is the most important sialic acid, the precursor of other sialic acid synthesis, and human The health relationship is most closely related; Neu5Ac can be used to synthesize antiviral drugs and to treat diseases such as H1N1 and H5N1 influenza. In addition, Neu5Ac also has great nutritional value. It can promote the development of the brain in infants, and also have a positive effect on the maintenance of brain function and health in premature infants. At present, the method of preparing and producing Neu5Ac is enzyme catalysis, which is a common method. Although the yield of the method is high, it needs to add excess pyruvic acid to lead to waste of raw materials, difficult product purification and environmental pressure. This paper attempts to establish a Neu5Ac synthesis method without pyruvic acid: N- acetyl glucosamine (Glc NAc) as the substrate and the N- acetyl glucosamine isomerase (AGE) catalyzed isomerization to N- acetyl mannose Amines (Man NAc); then, under the catalysis of Neu5Ac synthetase (Neu B), an irreversible reaction with phosphoenolpyruvic acid (PEP) is used to synthesize Neu5Ac. in Escherichia coli to construct the above-mentioned Neu5Ac synthesis pathway, and to strengthen the aggregation of Neu5Ac by metabolic engineering and fermentation engineering. The main conclusions are as follows: (1) examine AGE and Neu B from different sources, respectively. The results showed that the stability of B AGE in P AGE and colipora colipora was similar in the pig kidney, and the temperature preference and P H preference were similar. The optimum P H was 7, the optimum temperature was 50 degrees C, and the high activity was maintained in the 35-55 degree C peri, which reached 90 of its activity at 50 degree C. Under the condition of 37 C Tris-HCl (P H 7), P AGE and B AGE activity are 392.1 and 2411.5 U mg-1 total protein respectively. And the total protein of 6.380 U? Mg-1. But the stability of C Neu B was not good, only 50% of the catalytic activity was retained after 2 h. When co expression with the molecular chaperone P Gro7, the B activity of C Neu was 17.8% higher than that of the control group. Enterobacteriaceae expressed the ability to express C Neu B. Therefore, we consider the construction of Neu5Ac synthesis pathway based on the two enzymes of B AGE and C Neu B. (2) the synthesis pathway is constructed based on B AGE and C. Therefore, we focus on the catalytic activity of C Neu B in the system. A co expression vector p DTrc-AB and P DTrc-BA are obtained in order to adjust the two genes. It is found that cneu B gene in the P DTrc-AB is more active downstream, and the accumulation of the Neu5Ac decomposition pathway in the host Escherichia coli can not be realized. After the Nan ATEK gene cluster, E.coli SA-01 was obtained, and its Neu5Ac decomposition pathway was blocked; the co expression vector p DTrc-AB and P DTrc-BA were converted to E.coli SA-01. The induced organisms were successfully synthesized, and the synthesis amount was 2.61 and 2.03 respectively. Ineffective consumption, increasing the concentration of intracellular substrate Glc NAc and PEP supply, enhanced the synthesis of Neu5Ac, increased the synthesis of Neu5Ac by 38.3%, reached 3.61 G? L-1. (3), by which PEP synthesis related genes in Escherichia coli were expressed individually or together, improving the supply of intracellular PEP and further promoting the synthesis of Neu5Ac. Related genes PCK and PPS A, using Duet series vectors to construct a single overexpression and co expression vector of two genes, and import DE3 soluble E.coli. when PCK and PPS A are expressed separately, the Neu5Ac synthesis is 5.80 and 7.09 g, and the transcriptional level of the two genes increases synchronously and is positively correlated. The number of copies of the carrier. The optimal PEP fortified supply system is composed of medium copy carrier P CDF-pck-pps A. At this time, the Neu5Ac synthesis amount to 8.63 G? L-1, increase 139%. (4) compared with the starting strain, investigate the nutritional conditions of the synthesis of Neu5Ac and the effect of surfactant on Neu5Ac, optimize the Neu5Ac synthesis process, and in the fermenting tank. To achieve high efficiency synthesis of Neu5Ac. In the medium containing nitrogen sources, the host Escherichia coli grew in large quantities, and the synthesis of Neu5Ac was inhibited. In the nitrogen free glucose medium, the growth of Escherichia coli in the host was restricted, and the synthesis of Neu5Ac was inhibited and the synthesis reached 8.61 G? L-1. when the host Escherichia coli grew exuberant PEP and N The synthesis of PEP in the competitive cell of eu5Ac is not conducive to the synthesis of Neu5Ac. When glycerol is a carbon source, its transmembrane transport is not dependent on PTS, which saves the intracellular PEP and makes it more flow to the direction of Neu5Ac synthesis. The Neu5Ac synthesis also increases to the 10.43 G? L-1. conversion medium, adding Triton X-100, but has a certain promotion effect on the Neu5Ac synthesis. In the end of 6.1%., the process of biosynthesis of Neu5Ac in the fermenting tank was carried out. The phosphate concentration in the medium was greatly reduced and the permeation pressure of the reaction system was reduced. At the same time, the better mass transfer in the fermenting tank was beneficial to the synthesis of Neu5Ac. In the fermenting tank, the 65 h synthesis Neu5Ac reached 16.02 G? L-1, which was significantly increased by 53.6% than that of the same condition shake flask. The efficient synthesis of Neu5Ac is realized.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ920.1
【参考文献】
相关期刊论文 前1条
1 ;Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum[J];World Journal of Gastroenterology;2003年02期
，本文编号：2129334
本文链接：https://www.wllwen.com/shoufeilunwen/gckjbs/2129334.html
